CN111066654A - Tissue culture rapid propagation method of succulent plants - Google Patents
Tissue culture rapid propagation method of succulent plants Download PDFInfo
- Publication number
- CN111066654A CN111066654A CN201911263100.2A CN201911263100A CN111066654A CN 111066654 A CN111066654 A CN 111066654A CN 201911263100 A CN201911263100 A CN 201911263100A CN 111066654 A CN111066654 A CN 111066654A
- Authority
- CN
- China
- Prior art keywords
- culture
- percent
- explants
- medium
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000035755 proliferation Effects 0.000 claims abstract description 34
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 230000006698 induction Effects 0.000 claims abstract description 22
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 8
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 7
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 238000005286 illumination Methods 0.000 claims description 30
- 238000005406 washing Methods 0.000 claims description 30
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 19
- 229920001817 Agar Polymers 0.000 claims description 16
- 239000008272 agar Substances 0.000 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 230000003203 everyday effect Effects 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 238000002791 soaking Methods 0.000 claims description 15
- 229960004793 sucrose Drugs 0.000 claims description 15
- 239000005720 sucrose Substances 0.000 claims description 13
- 235000013343 vitamin Nutrition 0.000 claims description 13
- 239000011782 vitamin Substances 0.000 claims description 13
- 229940088594 vitamin Drugs 0.000 claims description 13
- 229930003231 vitamin Natural products 0.000 claims description 13
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000008223 sterile water Substances 0.000 claims description 10
- 239000008399 tap water Substances 0.000 claims description 10
- 235000020679 tap water Nutrition 0.000 claims description 10
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000012882 rooting medium Substances 0.000 claims description 7
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 6
- 229960001471 sodium selenite Drugs 0.000 claims description 6
- 235000015921 sodium selenite Nutrition 0.000 claims description 6
- 239000011781 sodium selenite Substances 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 230000035784 germination Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 3
- 230000000644 propagated effect Effects 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 abstract 7
- 238000000605 extraction Methods 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 13
- 239000000306 component Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 8
- 230000001737 promoting effect Effects 0.000 description 7
- 102000018997 Growth Hormone Human genes 0.000 description 4
- 108010051696 Growth Hormone Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000122 growth hormone Substances 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 3
- 239000004062 cytokinin Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000004161 plant tissue culture Methods 0.000 description 3
- LUBJCRLGQSPQNN-UHFFFAOYSA-N 1-Phenylurea Chemical compound NC(=O)NC1=CC=CC=C1 LUBJCRLGQSPQNN-UHFFFAOYSA-N 0.000 description 2
- HZFDKBPTVOENNB-GAFUQQFSSA-N N-[(2S)-1-[2-[(2R)-2-chloro-2-fluoroacetyl]-2-[[(3S)-2-oxopyrrolidin-3-yl]methyl]hydrazinyl]-3-(1-methylcyclopropyl)-1-oxopropan-2-yl]-5-(difluoromethyl)-1,2-oxazole-3-carboxamide Chemical compound CC1(C[C@@H](C(NN(C[C@H](CCN2)C2=O)C([C@H](F)Cl)=O)=O)NC(C2=NOC(C(F)F)=C2)=O)CC1 HZFDKBPTVOENNB-GAFUQQFSSA-N 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000005648 plant growth regulator Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 description 1
- 239000005979 Forchlorfenuron Substances 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- GPXLRLUVLMHHIK-UHFFFAOYSA-N forchlorfenuron Chemical group C1=NC(Cl)=CC(NC(=O)NC=2C=CC=CC=2)=C1 GPXLRLUVLMHHIK-UHFFFAOYSA-N 0.000 description 1
- 230000005089 fruit drop Effects 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003898 horticulture Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of succulent plant planting, in particular to a tissue culture and rapid propagation method of succulent plants. The extraction method comprises the following steps: the method comprises the following steps: s1) explant selection and sterilization: s2) primary induction culture: s3) proliferation culture: s4) inducing rooting culture: s5) hardening off and transplanting. Also discloses the components and the proportion of the primary induction culture medium, the multiplication culture medium and the rooting culture medium. The succulent plant seedlings obtained by the tissue culture and rapid propagation method have short production period, high propagation frequency and regular seedlings, the propagation coefficient and propagation speed of the succulent plants are improved, the germination rate and the rooting rate of the succulent plant seedlings are about 3 times of those of a conventional culture medium, the strain advantages of the succulent plants can be well kept, a large number of tissue culture seedlings suitable for transplanting can be rapidly propagated, the grown seedling plants are consistent in shape, the operation is easy, and the industrial production is facilitated.
Description
Technical Field
The invention relates to the technical field of succulent plant planting, in particular to a tissue culture and rapid propagation method of succulent plants.
Background
Succulent plants are plants that have developed parenchyma to store water and appear thick and juicy in appearance, and are part of the vegetative organs of plants. In horticulture, the plants are also called succulent plants or succulent flowers, but succulent plants are most commonly used. In recent years, with the help of the powerful propaganda of the internet and the characteristics of low maintenance starting point, relatively obvious and stable appearance, gorgeous and various colors and the like of succulents, the succulents are well known and loved by the public in a short time and are then popular nationwide. At present, the succulent plant propagation technology mostly adopts leaf cutting propagation, and has the advantages of low efficiency, low propagation coefficient, long propagation period, field requirement and high labor cost. Therefore, the problems to be solved at present are to improve the production efficiency of succulent plants, shorten the cultivation time and reduce the labor cost.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a tissue culture rapid propagation method of succulent plants with reliable performance, and solves the problems of low propagation coefficient and long propagation period of the traditional succulent plants.
The technical scheme of the invention is realized as follows: a tissue culture and rapid propagation method of succulent plants is characterized in that: the method comprises the following steps:
s1) explant selection and sterilization:
taking leaves or stems of succulent plants as explants, putting the explants in a beaker, washing the explants cleanly with tap water, washing the explants with a proper amount of detergent, washing the washed explants with the tap water for more than 3min, soaking the explants in 75% alcohol for 30s on a superclean bench, washing the washed explants with sterile water for 2-3 times, quickly pouring the washed explants into 0.1% mercuric chloride solution for soaking for 5min, taking out the explants, washing the explants with the sterile water for 4-5 times, and sucking the water with filter paper to obtain sterile explants;
s2) primary induction culture:
under the aseptic condition, cutting the obtained explant into small slices with the length of 1-2 cm, soaking the small slices in 75% alcohol, washing with water, and then respectively inoculating the small slices into primary induction culture media for culture, wherein the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 55-65%, the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, and the culture time is 15-20 d until the explant is differentiated into cluster buds;
s3) proliferation culture:
transferring the obtained cluster buds into a proliferation culture medium under an aseptic condition for proliferation culture until the length of the cluster buds is 3-5 cm and the proliferation coefficient is 3-5 times, wherein the culture condition is as follows: the temperature is 27-32 ℃, the air humidity is 55-65%, the illumination intensity is 1200-1600 lux, the illumination is 12-16 h every day, and the culture time is 30-40 d;
s4) inducing rooting culture:
when the leaves grow to 1-1.5 cm, taking out the seedlings, and inoculating the seedlings into a rooting culture medium in a culture bottle under an aseptic condition for continuous culture; the culture conditions were: the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, the temperature is 18-23 ℃, and the culture time is 5-7 d;
s5) hardening off and transplanting:
after rooting or the root system is basically developed, moving the culture bottle to the outdoor, performing closed bottle seedling hardening for 7-10 days with shading degree of 50-70%, then performing open bottle seedling hardening for 2-3 days under natural light to obtain a complete plant, and outplanting.
Preferably, the primary induction medium in the S2 comprises the following components: by mass percentage, 3 to 5 percent of sucrose, 0.8 to 1.3 percent of agar, 0.01 to 0.02 percent of sodium selenite, 0.2 to 0.4 percent of MS +6-BA, 0.2 to 0.5 percent of NAA, 0.08 to 0.12 percent of IBA, 0.015 to 0.03 percent of vitamin complex and the balance of water.
Preferably, the proliferation medium in S3 comprises: according to mass percentage, 3 to 6 percent of sucrose, 0.8 to 1.3 percent of agar, 0.01 to 0.02 percent of KT, 0.2 to 0.6 percent of MS +6-BA, 0.2 to 0.4 percent of NAA, 30.08 to 0.12 percent of GA, 0.015 to 0.03 percent of vitamin complex and the balance of water.
Preferably, the rooting medium in the S4 comprises the following components: according to mass percentage, 2 to 5 percent of cane sugar, 0.6 to 1.2 percent of agar, 0.2 to 0.5 percent of MS +6-BA, 0.2 to 0.5 percent of NAA, 30.08 to 0.12 percent of GA and the balance of water.
The pH values of the primary induction culture medium, the multiplication culture medium and the rooting culture medium are 6.3-7.
The invention solves the defects in the background technology and has the following beneficial effects:
aiming at the defects of the prior art, the invention solves the problems of low propagation coefficient and long propagation period of the traditional succulent plants. The invention provides a tissue culture and rapid propagation method of succulent plants, which comprises the steps of explant selection and disinfection, primary generation induction culture, proliferation culture, induced rooting culture, seedling hardening and transplanting. The culture medium prepared by adopting the growth hormone has short production period, high proliferation frequency and regular seedlings, improves the propagation coefficient and the propagation speed of succulent plants, has the germination rate and the rooting rate about 3 times of those of the conventional culture medium, can better maintain the strain advantages of the succulent plants, and can rapidly propagate a large amount of tissue culture seedlings suitable for transplantation. And the grown seedling plants have the same shape, are easy to operate and are convenient for industrial production.
Detailed Description
Example 1
The tissue culture and rapid propagation method of succulent plants is characterized in that: the method comprises the following steps:
s1) explant selection and sterilization:
taking leaves or stems of succulent plants as explants, putting the explants in a beaker, washing the explants cleanly with tap water, washing the explants with a proper amount of detergent, washing the washed explants with the tap water for more than 3min, soaking the explants in 75% alcohol for 30s on a superclean bench, washing the washed explants with sterile water for 2-3 times, quickly pouring the washed explants into 0.1% mercuric chloride solution for soaking for 5min, taking out the explants, washing the explants with the sterile water for 4-5 times, and sucking the water with filter paper to obtain sterile explants;
s2) primary induction culture:
under the aseptic condition, cutting the obtained explant into small slices with the length of 1-2 cm, soaking the small slices in 75% alcohol, washing with water, and then respectively inoculating the small slices into primary induction culture media for culture, wherein the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 55-65%, the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, and the culture time is 15-20 d until the explant is differentiated into cluster buds; the primary induction medium comprises the following components: by mass percentage, 4 percent of sucrose, 1.0 percent of agar, 0.015 percent of sodium selenite, 0.3 percent of MS +6-BA, 0.3 percent of NAA, 0.10 percent of IBA, 0.02 percent of vitamin complex and the balance of water.
S3) proliferation culture:
transferring the obtained cluster buds into a proliferation culture medium under an aseptic condition for proliferation culture until the length of the cluster buds is 3-5 cm and the proliferation coefficient is 3-5 times, wherein the culture condition is as follows: the temperature is 27-32 ℃, the air humidity is 55-65%, the illumination intensity is 1200-1600 lux, the illumination is 12-16 h every day, and the culture time is 30-40 d; the proliferation medium in S3 consists of: by mass percentage, 4.5 percent of sucrose, 1.1 percent of agar, 0.016 percent of KT, 0.5 percent of MS +6-BA, 0.3 percent of NAA, 30.1 percent of GA, 0.02 percent of vitamin complex and the balance of water.
S4) inducing rooting culture:
when the leaves grow to 1-1.5 cm, taking out the seedlings, and inoculating the seedlings into a rooting culture medium in a culture bottle under an aseptic condition for continuous culture; the culture conditions were: the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, the temperature is 18-23 ℃, and the culture time is 5-7 d; the rooting medium comprises the following components: according to mass percentage, 4 percent of sucrose, 0.9 percent of agar, 0.3 percent of MS +6-BA, 0.4 percent of NAA, 30.10 percent of GA and the balance of water.
S5) hardening off and transplanting:
after rooting or the root system is basically developed, moving the culture bottle to the outdoor, performing closed bottle seedling hardening for 7-10 days with shading degree of 50-70%, then performing open bottle seedling hardening for 2-3 days under natural light to obtain a complete plant, and outplanting.
Example 2
The tissue culture and rapid propagation method of succulent plants is characterized in that: the method comprises the following steps:
s1) explant selection and sterilization:
taking leaves or stems of succulent plants as explants, putting the explants in a beaker, washing the explants cleanly with tap water, washing the explants with a proper amount of detergent, washing the washed explants with the tap water for more than 3min, soaking the explants in 75% alcohol for 30s on a superclean bench, washing the washed explants with sterile water for 2-3 times, quickly pouring the washed explants into 0.1% mercuric chloride solution for soaking for 5min, taking out the explants, washing the explants with the sterile water for 4-5 times, and sucking the water with filter paper to obtain sterile explants;
s2) primary induction culture:
under the aseptic condition, cutting the obtained explant into small slices with the length of 1-2 cm, soaking the small slices in 75% alcohol, washing with water, and then respectively inoculating the small slices into primary induction culture media for culture, wherein the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 55-65%, the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, and the culture time is 15-20 d until the explant is differentiated into cluster buds; the primary induction medium comprises the following components: by mass percentage, 3 percent of sucrose, 0.8 percent of agar, 0.01 percent of sodium selenite, 0.2 percent of MS +6-BA, 0.2 percent of NAA, 0.08 percent of IBA, 0.015 percent of vitamin complex and the balance of water.
S3) proliferation culture:
transferring the obtained cluster buds into a proliferation culture medium under an aseptic condition for proliferation culture until the length of the cluster buds is 3-5 cm and the proliferation coefficient is 3-5 times, wherein the culture condition is as follows: the temperature is 27-32 ℃, the air humidity is 55-65%, the illumination intensity is 1200-1600 lux, the illumination is 12-16 h every day, and the culture time is 30-40 d; the proliferation medium in S3 consists of: according to mass percentage, 3 percent of sucrose, 0.8 percent of agar, 0.01 percent of KT, 0.2 percent of MS +6-BA, 0.2 percent of NAA, 30.08 percent of GA, 0.015 percent of vitamin complex and the balance of water.
S4) inducing rooting culture:
when the leaves grow to 1-1.5 cm, taking out the seedlings, and inoculating the seedlings into a rooting culture medium in a culture bottle under an aseptic condition for continuous culture; the culture conditions were: the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, the temperature is 18-23 ℃, and the culture time is 5-7 d; the rooting medium comprises the following components: according to mass percentage, 2 percent of sucrose, 0.6 percent of agar, 0.2 percent of MS +6-BA, 0.2 percent of NAA, 30.08 percent of GA and the balance of water.
S5) hardening off and transplanting:
after rooting or the root system is basically developed, moving the culture bottle to the outdoor, performing closed bottle seedling hardening for 7-10 days with shading degree of 50-70%, then performing open bottle seedling hardening for 2-3 days under natural light to obtain a complete plant, and outplanting.
Example 3
The tissue culture and rapid propagation method of succulent plants is characterized in that: the method comprises the following steps:
s1) explant selection and sterilization:
taking leaves or stems of succulent plants as explants, putting the explants in a beaker, washing the explants cleanly with tap water, washing the explants with a proper amount of detergent, washing the washed explants with the tap water for more than 3min, soaking the explants in 75% alcohol for 30s on a superclean bench, washing the washed explants with sterile water for 2-3 times, quickly pouring the washed explants into 0.1% mercuric chloride solution for soaking for 5min, taking out the explants, washing the explants with the sterile water for 4-5 times, and sucking the water with filter paper to obtain sterile explants;
s2) primary induction culture:
under the aseptic condition, cutting the obtained explant into small slices with the length of 1-2 cm, soaking the small slices in 75% alcohol, washing with water, and then respectively inoculating the small slices into primary induction culture media for culture, wherein the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 55-65%, the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, and the culture time is 15-20 d until the explant is differentiated into cluster buds; the primary induction medium comprises the following components: according to the mass percentage, the health-care food comprises 5 percent of sucrose, 1.3 percent of agar, 0.02 percent of sodium selenite, 0.4 percent of MS +6-BA, 0.5 percent of NAA, 0.12 percent of IBA, 0.03 percent of vitamin complex and the balance of water.
S3) proliferation culture:
transferring the obtained cluster buds into a proliferation culture medium under an aseptic condition for proliferation culture until the length of the cluster buds is 3-5 cm and the proliferation coefficient is 3-5 times, wherein the culture condition is as follows: the temperature is 27-32 ℃, the air humidity is 55-65%, the illumination intensity is 1200-1600 lux, the illumination is 12-16 h every day, and the culture time is 30-40 d; the proliferation medium in S3 consists of: according to the mass percentage, the sugar is 6 percent, the agar is 1.3 percent, the KT is 0.02 percent, the MS +6-BA is 0.6 percent, the NAA is 0.4 percent, the GA is 30.12 percent, the vitamin complex is 0.03 percent, and the balance is water.
S4) inducing rooting culture:
when the leaves grow to 1-1.5 cm, taking out the seedlings, and inoculating the seedlings into a rooting culture medium in a culture bottle under an aseptic condition for continuous culture; the culture conditions were: the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, the temperature is 18-23 ℃, and the culture time is 5-7 d; the rooting medium comprises the following components: according to mass percentage, 5 percent of sucrose, 1.2 percent of agar, 0.5 percent of MS +6-BA, 0.5 percent of NAA, 30.12 percent of GA and the balance of water.
S5) hardening off and transplanting:
after rooting or the root system is basically developed, moving the culture bottle to the outdoor, performing closed bottle seedling hardening for 7-10 days with shading degree of 50-70%, then performing open bottle seedling hardening for 2-3 days under natural light to obtain a complete plant, and outplanting.
Comparative example
The culture procedure is the same as the above example, except that a single culture medium is used in the culture process, and the culture medium components are as follows: by mass percentage, 4 percent of sucrose, 1.0 percent of agar, 0.015 percent of sodium selenite, 0.3 percent of MS +6-BA, 0.3 percent of NAA and the balance of water. The culture conditions in the medium were observed and recorded, and the specific grouping and culture results are shown in the following table.
TABLE 1 Effect of the respective major ingredients and concentrations in the Primary Induction Medium on the differentiation of succulent plants
| Group of | IBA％ | Composite vitamin% | Number of inoculation | Number of differentiation | Rate of differentiation |
| Example 1 | 0.10 | 0.02 | 100 | 95 | 95％ |
| Example 2 | 0.08 | 0.015 | 100 | 90 | 90％ |
| Example 3 | 0.12 | 0.03 | 100 | 88 | 88％ |
| Comparative example | -- | -- | 100 | 65 | 65％ |
From Table 1, it is apparent that by comparing example 1, example 2, example 3 and comparative example 1 of the present invention, the differentiation rate was much higher than that of the comparative example after the simultaneous administration of IBA and multivitamin, and the highest differentiation rate was observed when IBA was 0.1% and vitamin complex was 0.02%.
TABLE 2 Effect of the respective major ingredients and concentrations in the multiplication Medium on the multiplication of succulent plants
| Group of | KT％ | GA3％ | Composite vitamin% | Number of inoculation | Number of proliferations | Proliferation rate |
| Example 1 | 0.016 | 0.10 | 0.02 | 100 | 92 | 92％ |
| Example 2 | 0.01 | 0.08 | 0.015 | 100 | 80 | 80％ |
| Example 3 | 0.02 | 0.12 | 0.03 | 100 | 85 | 85％ |
| Comparative example | -- | -- | -- | 100 | 20 | 20％ |
As is apparent from table 2 comparing example 1, example 2, example 3 and comparative example 1 of the present invention, the proliferation rate was significantly increased after the simultaneous administration of KT, GA3 and multivitamin, and the highest proliferation rate was observed when KT was 0.016%, GA was 30.10%, and multivitamin was 0.02%.
TABLE 3 Effect of the respective major ingredients and concentrations in rooting Medium on the differentiation of succulent plants
| Group of | GA3％ | Number of inoculation | Root number of root | Rooting rate |
| Example 1 | 0.10 | 100 | 93 | 93％ |
| Example 2 | 0.08 | 100 | 86 | 86％ |
| Example 3 | 0.12 | 100 | 81 | 81％ |
| Comparative example | -- | 2 | 2％ |
From table 3, it is apparent from comparison of example 1, example 2, example 3 and comparative example 1 of the present invention that the rooting rate is significantly increased after GA3 is applied, and the rooting rate is highest when GA is 30.10%.
The MS +6-BA of the invention is that 6-BA is added into an MS basic culture medium to be 6-benzylamino adenine, is a plant growth hormone, has the main functions of promoting the formation of buds, inducing callus to generate, promoting cell division, promoting the differentiation of non-differentiated tissues, promoting the accumulation of substances in organisms, promoting the generation of lateral buds and preventing aging, and is a cytokinin in plant tissues and cell culture. NAA is naphthylacetic acid, is a plant growth hormone, is used when plants are propagated by a cutting method, can also be used for plant tissue culture, can promote cell division and expansion, induce to form adventitious roots to increase fruit setting, prevent fruit drop, change the ratio of female flowers and male flowers and the like, can enter the plants through the tender epidermis of leaves and branches, and seeds enter the plants to be guided to the whole plants along with nutrient flow. KT is forchlorfenuron, is a phenylurea plant growth regulator with cytokinin activity, and has the biological activity 10-100 times higher than that of 6-benzylaminopurine. It can affect the development of plant bud, accelerate cell mitosis, promote cell enlargement and differentiation, prevent fruit and flower from dropping, and can also be used as cytokinin for plant tissue culture. GA3 is gibberellin, is a plant growth regulator, and is mainly used for promoting the growth and development of crops, maturing in advance, increasing yield, breaking dormancy of organs such as seeds, tubers and bulbs, promoting germination, tillering and bolting, increasing fruit bearing rate, and can also be used for plant tissue culture.
The invention provides a tissue culture and rapid propagation method of succulent plants, which comprises the steps of explant selection and disinfection, primary generation induction culture, proliferation culture, induced rooting culture, seedling hardening and transplanting. The culture medium prepared by adopting the growth hormone has short production period, high proliferation frequency and regular seedlings, improves the propagation coefficient and the propagation speed of succulent plants, has the germination rate and the rooting rate about 3 times of those of the conventional culture medium, can better maintain the strain advantages of the succulent plants, and can rapidly propagate a large amount of tissue culture seedlings suitable for transplantation. And the grown seedling plants have the same shape, are easy to operate and are convenient for industrial production.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A tissue culture and rapid propagation method of succulent plants is characterized in that: the method comprises the following steps:
s1) explant selection and sterilization:
taking leaves or stems of succulent plants as explants, putting the explants in a beaker, washing the explants cleanly with tap water, washing the explants with a proper amount of detergent, washing the washed explants with the tap water for more than 3min, soaking the explants in 75% alcohol for 30s on a superclean bench, washing the washed explants with sterile water for 2-3 times, quickly pouring the washed explants into 0.1% mercuric chloride solution for soaking for 5min, taking out the explants, washing the explants with the sterile water for 4-5 times, and sucking the water with filter paper to obtain sterile explants;
s2) primary induction culture:
under the aseptic condition, cutting the obtained explant into small slices with the length of 1-2 cm, soaking the small slices in 75% alcohol, washing with water, and then respectively inoculating the small slices into primary induction culture media for culture, wherein the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 55-65%, the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, and the culture time is 15-20 d until the explant is differentiated into cluster buds;
s3) proliferation culture:
transferring the obtained cluster buds into a proliferation culture medium under an aseptic condition for proliferation culture until the length of the cluster buds is 3-5 cm and the proliferation coefficient is 3-5 times, wherein the culture condition is as follows: the temperature is 27-32 ℃, the air humidity is 55-65%, the illumination intensity is 1200-1600 lux, the illumination is 12-16 h every day, and the culture time is 30-40 d;
s4) inducing rooting culture:
when the leaves grow to 1-1.5 cm, taking out the seedlings, and inoculating the seedlings into a rooting culture medium in a culture bottle under an aseptic condition for continuous culture; the culture conditions were: the illumination intensity is 1600-2000 lux, the illumination is 12-16 h every day, the temperature is 18-23 ℃, and the culture time is 5-7 d;
s5) hardening off and transplanting:
after rooting or the root system is basically developed, moving the culture bottle to the outdoor, performing closed bottle seedling hardening for 7-10 days with shading degree of 50-70%, then performing open bottle seedling hardening for 2-3 days under natural light to obtain a complete plant, and outplanting.
2. The tissue culture and rapid propagation method of succulent plants according to claim 1, wherein the primary induction medium in S2 comprises: by mass percentage, 3 to 5 percent of sucrose, 0.8 to 1.3 percent of agar, 0.01 to 0.02 percent of sodium selenite, 0.2 to 0.4 percent of MS +6-BA, 0.2 to 0.5 percent of NAA, 0.08 to 0.12 percent of IBA, 0.015 to 0.03 percent of vitamin complex and the balance of water.
3. The tissue culture and rapid propagation method of succulent plants according to claim 1, wherein the propagation medium in S3 comprises: according to mass percentage, 3 to 6 percent of sucrose, 0.8 to 1.3 percent of agar, 0.01 to 0.02 percent of KT, 0.2 to 0.6 percent of MS +6-BA, 0.2 to 0.4 percent of NAA, 30.08 to 0.12 percent of GA, 0.015 to 0.03 percent of vitamin complex and the balance of water.
4. The tissue culture and rapid propagation method of succulent plants according to claim 1, wherein the rooting medium in S4 comprises: according to mass percentage, 2 to 5 percent of cane sugar, 0.6 to 1.2 percent of agar, 0.2 to 0.5 percent of MS +6-BA, 0.2 to 0.5 percent of NAA, 30.08 to 0.12 percent of GA and the balance of water.
5. The tissue culture and rapid propagation method of succulent plants according to claim 1, wherein the pH values of the primary induction medium, the proliferation medium and the rooting medium are 6.3-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911263100.2A CN111066654A (en) | 2019-12-11 | 2019-12-11 | Tissue culture rapid propagation method of succulent plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911263100.2A CN111066654A (en) | 2019-12-11 | 2019-12-11 | Tissue culture rapid propagation method of succulent plants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111066654A true CN111066654A (en) | 2020-04-28 |
Family
ID=70313698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911263100.2A Pending CN111066654A (en) | 2019-12-11 | 2019-12-11 | Tissue culture rapid propagation method of succulent plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111066654A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113133409A (en) * | 2021-06-04 | 2021-07-20 | 福建三杉生物科技有限公司 | Tissue culture process of seven fushen |
| CN113826553A (en) * | 2021-10-28 | 2021-12-24 | 江苏农林职业技术学院 | Method for culturing and rapidly propagating succulent plant Tianzhuang pancakea tissues |
| CN114223542A (en) * | 2021-12-22 | 2022-03-25 | 山西省农业科学院园艺研究所 | Succulent plant tissue culture rapid propagation method based on explants |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080261310A1 (en) * | 2007-04-20 | 2008-10-23 | Conopco, Inc., D/B/A/ Unilever | In vitro rooting of hoodia plants |
| CN102499090A (en) * | 2011-11-07 | 2012-06-20 | 上海旭东园艺有限公司 | Method for isolated culture of Haworthia succulent plants |
| CN104273028A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院上海生命科学研究院 | Method for rapid in-vitro propagation of Crassulaceae plant |
| CN105638463A (en) * | 2015-12-30 | 2016-06-08 | 四川禾木本业农林科技有限公司 | Tissue-culture rapid propagation method for succulents |
| CN105766635A (en) * | 2016-03-14 | 2016-07-20 | 龙岩市禾康生物科技有限公司 | Method for tissue culture and rapid propagation of succulent plants |
| CN105850747A (en) * | 2016-05-13 | 2016-08-17 | 大连民族大学 | Rapid propagation method for tissue of succulent sedum rubrotinctum and sedum rubrotinctum cultured with method |
| CN105961198A (en) * | 2013-09-16 | 2016-09-28 | 周杰 | Tissue culture method of dendrobium officinale |
| CN108308027A (en) * | 2018-03-15 | 2018-07-24 | 广西壮族自治区亚热带作物研究所 | A kind of method for tissue culture in succulent moonlight longevity |
-
2019
- 2019-12-11 CN CN201911263100.2A patent/CN111066654A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080261310A1 (en) * | 2007-04-20 | 2008-10-23 | Conopco, Inc., D/B/A/ Unilever | In vitro rooting of hoodia plants |
| WO2008128842A1 (en) * | 2007-04-20 | 2008-10-30 | Unilever N.V. | In vitro rooting of hoodia plants |
| CN102499090A (en) * | 2011-11-07 | 2012-06-20 | 上海旭东园艺有限公司 | Method for isolated culture of Haworthia succulent plants |
| CN104273028A (en) * | 2013-07-03 | 2015-01-14 | 中国科学院上海生命科学研究院 | Method for rapid in-vitro propagation of Crassulaceae plant |
| CN105961198A (en) * | 2013-09-16 | 2016-09-28 | 周杰 | Tissue culture method of dendrobium officinale |
| CN105638463A (en) * | 2015-12-30 | 2016-06-08 | 四川禾木本业农林科技有限公司 | Tissue-culture rapid propagation method for succulents |
| CN105766635A (en) * | 2016-03-14 | 2016-07-20 | 龙岩市禾康生物科技有限公司 | Method for tissue culture and rapid propagation of succulent plants |
| CN105850747A (en) * | 2016-05-13 | 2016-08-17 | 大连民族大学 | Rapid propagation method for tissue of succulent sedum rubrotinctum and sedum rubrotinctum cultured with method |
| CN108308027A (en) * | 2018-03-15 | 2018-07-24 | 广西壮族自治区亚热带作物研究所 | A kind of method for tissue culture in succulent moonlight longevity |
Non-Patent Citations (2)
| Title |
|---|
| 李小东: "多肉植物组培技术", 《中国花卉园艺》 * |
| 金燕等: "多肉类十二卷属植物组织培养技术", 《上海农业科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113133409A (en) * | 2021-06-04 | 2021-07-20 | 福建三杉生物科技有限公司 | Tissue culture process of seven fushen |
| CN113133409B (en) * | 2021-06-04 | 2022-04-19 | 福建三杉生物科技有限公司 | Tissue culture process of seven fushen |
| CN113826553A (en) * | 2021-10-28 | 2021-12-24 | 江苏农林职业技术学院 | Method for culturing and rapidly propagating succulent plant Tianzhuang pancakea tissues |
| CN114223542A (en) * | 2021-12-22 | 2022-03-25 | 山西省农业科学院园艺研究所 | Succulent plant tissue culture rapid propagation method based on explants |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103931497B (en) | A kind of method improving dragon fruit plantlet in vitro planting percent | |
| CN103563748B (en) | A kind of method of cultivating Virus-free Tube Potato Plantlets strong sprout | |
| CN111066654A (en) | Tissue culture rapid propagation method of succulent plants | |
| CN102845313A (en) | Method for quickly in-vitro actinidia kolomikta propagating | |
| CN113826550B (en) | Somatic embryogenesis and tissue culture method for camphor trees | |
| CN104160956A (en) | High efficient and fast culture method of anoectochilus roxburghii tissue cultured seedling | |
| CN104221859B (en) | A kind of fast numerous cultural method of Garcinia mangostana | |
| CN105284620A (en) | Method for rescuing grown-up seedlings of special precocious peach hybrid embryos | |
| CN108077070B (en) | Culture medium and culture method for maple tissue culture | |
| CN104106468A (en) | Tissue culture and rapid propagation method of radix fici simplicissimae | |
| CN105638463A (en) | Tissue-culture rapid propagation method for succulents | |
| CN108029559B (en) | Method for quickly cultivating ilex latifolia tissue culture seedlings | |
| CN112715367B (en) | Method for carrying out Maozu secondary proliferation by using lanthanum nitrate | |
| CN102138532A (en) | Technique for breeding seedlings of Taihe tribute Chinese toon by tissue culture | |
| CN101185421B (en) | Method of cultivating plant by 'hanfu' apple anther | |
| CN102577972A (en) | Method for tissue culture of hoya kerrii | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN108849500B (en) | Culture medium and method for lotus embryo rescue and rapid growth and development of offspring | |
| CN114431154B (en) | Method for asexual propagation through acer nikoense dormant buds | |
| David et al. | Preliminary results on the influence of growth hormones on the in vitro regeneration of Phalaenopsis flower stalks. | |
| CN104737919A (en) | Tissue culture rapid seedlings growing method used for cherry stock M9 | |
| CN101707981A (en) | Rubber tree cotyledon embryo high-efficiency embryonic callus induction and regeneration method | |
| CN114424749A (en) | Liriope spicata in-vitro rapid propagation method | |
| CN114041421A (en) | Tissue rapid propagation method of avocados | |
| CN103202226A (en) | Quick and efficient rooting method for Chinese cabbage tissue culture seedlings |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200428 |
|
| RJ01 | Rejection of invention patent application after publication |